Reprogramming tumor-associated macrophages to outcompete endovascular endothelial progenitor cells and suppress tumor neoangiogenesis

Abstract

Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.

Keywords: TSC; cell competition; endothelial progenitor cell; mTORC1; tumor-associated macrophage.

Figure 1.. mTORC1 signaling in TAMs is lower than that in MTMs in mammary tumors

(A) Representative images (left) and quantification (right) of localization of Vcam1^hi tumor-associated macrophages (TAMs) and CD206^hi mammary tissue macrophages (MTMs) in PyMT tumor tissue. F4/80⁺ macrophages are indicated by yellow arrowheads. E-Cadherin⁺ region is defined as tumor and E-Cadherin⁻ region is counted as stroma. Data are pooled from two independent experiments with 5 randomly selected tumor regions for quantification. Scale bars: 200 μm, 50 μm, and 50 μm. (B) Representative images of Vcam1 (red), CD206 (cyan), F4/80 (white), pS6 (green), and DAPI staining in Vcam1^hi TAMs and CD206^hi MTMs in PyMT tumor tissue (left). Contour of individual macrophages are traced by dashed line. Quantification of average cell area and pS6 intensity of Vcam1^hi TAMs (n = 36) and CD206^hi MTMs (n = 52) (right). Scale bars: 5 μm and 5 μm. Unpaired t-test, two-tailed, ***: p < 0.001, ****: p < 0.0001. Data are shown as mean ± SEM. See also Figure S1.

Figure 2.. Loss of TSC1 results in mTORC1-dependent TAM reprogramming and tumor repression

(A) Representative images of Vcam1 (red), F4/80 (white), pS6 (green), and DAPI staining of Vcam1^hi TAMs in Tsc1^fl/flPyMT, CD11c^CreTsc1^fl/flPyMT, CD11c^CreRptor^fl/flPyMT and CD11c^CreTsc1^fl/flRptor^fl/flPyMT tumor tissues (left). Contour of individual macrophages are traced by dashed line. Quantification of average cell area and pS6 intensity of Vcam1^high TAMs (right). Number of Vcam1^high TAMs quantified for each genotype: Tsc1^fl/flPyMT (n = 38), CD11c^CreTsc1^fl/flPyMT (n = 34), CD11c^CreRptor^fl/flPyMT (n = 51) and CD11c^CreTsc1^fl/flRptor^fl/flPyMT (n = 45). Scale bars: 5 μm, 5 μm, 5 μm, and 5 μm. (B) Total tumor burden of Tsc1^fl/flPyMT (n = 48), CD11c^CreTsc1^fl/flPyMT (n = 56), CD11c^CreRptor^fl/flPyMT (n = 8) and CD11c^CreTsc1^fl/flRptor^fl/flPyMT (n = 17) mice. Unpaired t-test, two-tailed, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, and n.s. = not significant. Data are shown as mean ± SEM. T-test in (B) is calculated based on endpoint tumor burden. See also Figure S2.

Figure 3.. Increased cancer cell death and diminished tumor growth in mice with TSC1-deficient TAMs are independent of the adaptive immune system

(A) Representative image (left) and quantification (right) of Ki67 and cleaved Caspase 3 (CC3) expression in E-Cadherin⁺ cancer cells from randomly selected Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT tumor tissues (n = 6–16). Scale bars: 50 μm and 50 μm. (B) Representative image (left) and quantification (right) of CD4⁺ and CD8⁺ T cell localization from randomly selected Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT tumor tissues (n = 8–10). Scale bars: 50 μm and 50 μm. Intra-tumoral and stromal CD4⁺ T cells are indicated by magenta arrowheads and arrows, respectively. Intra-tumoral and stromal CD8⁺ T cells are indicated by yellow arrowheads and arrows, respectively. (C) Representative image (left) and quantification (right) of Ki67⁺ and CC3⁺ cancer cells from randomly selected CD11c^CreTsc1^fl/flRag1^−/−PyMT and Tsc1^fl/flRag1^−/−PyMT tumor regions (n = 6–12). Scale bars: 50 μm and 50 μm. (D) Total tumor burden of CD11c^CreTsc1^fl/flRag1^−/−PyMT (n = 6) and littermate Tsc1^fl/flRag1^−/−PyMT controls (n = 7). Unpaired t-test, two-tailed, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, and n.s. = not significant. Data are shown as mean ± SEM. T-test in (D) is calculated based on endpoint tumor burden. See also Figure S3.

Figure 4.. Loss of TSC1 triggers TAM repositioning to a perivascular niche in association with vessel reorganization, tumor tissue healing, hypoxia, and cancer cell death

(A) Representative images of tissue localization of F4/80⁺Vcam1^hi tumor-associated macrophages (TAMs) and F4/80⁺Vcam1^lo/− mammary tissue macrophages (MTMs) in tumor tissues of Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT mice (top). Orange arrows indicate TAMs or MTMs. Magenta arrows indicate cell death regions with condensed and fragmented chromatin. Quantification of the distance of individual TAMs to their nearest CD31⁺ vasculature (bottom). Data are representative of three independent experiments with more than 1×10³ TAMs quantified in each experiment. Scale bars: 200 μm, 200 μm, 50 μm, 50 μm, 50 μm, and 50 μm. (B) Representative images of tumor sections from Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT mice with CD31 (red), Fibrinogen (Fg, white), Cleaved Caspase 3 (cyan), E-Cadherin (green), and DAPI staining (left). Isolated CD31 staining is indicated by magenta arrows and extravascular Fg⁺ events are indicated by orange arrows. Quantification of isolated CD31 staining (top right) and extravascular Fg⁺ events (bottom right) in Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT tumor tissues per 1 mm² (n = 8-14). Scale bars: 200 μm, 50 μm, 200 μm, and 50 μm. (C) Representative images (left) and quantification (right) of pimonidazole-positive hypoxic area in Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT (n = 10 each). Scale bars: 50 μm and 50 μm. All data are shown as mean ± SEM, unpaired t-test, two-tailed, *: p < 0.05 and ****: p < 0.0001. See also Figure S4.

Figure 5.. TSC1-deficient TAMs exhibit an anti-inflammatory and pro-resolving cell differentiation state

(A) Uniform manifold approximation and projection (UMAP) of single cells in two-dimensions and clustered into 5 communities using the Leiden community detection (left). UMAP projection of single cells in two-dimensions in which tumor-associated macrophages (TAMs) from Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT were colored blue and purple respectively (middle). The fraction of TSC1-deficient TAMs and wild-type TAMs in each cluster (right). (B) Matrix plot showing the expression of representative genes related to different biological processes in cluster 1, cluster 2, and cluster 4. (*: genes encoding lysosomal proteins. See also Table S3.) See also Figure S5.

Figure 6.. TSC1-deficient TAMs display enhanced efferocytosis and mitochondrial activities

(A) Representative flow cytometric analysis (left) and quantification (right) of CD107a (LAMP1) expression in TAMs from Tsc1^fl/flPyMT (n = 7) and CD11c^CreTsc1^fl/flPyMT (n = 6) mice. (B) Representative flow cytometric analysis (left) and quantification (right) of CellTrace Violet (CTV)-labelled apoptotic cell engulfment in TAMs from Tsc1^fl/flPyMT (n = 3) and CD11c^CreTsc1^fl/flPyMT (n = 3) mice. (C) Representative flow cytometric analysis (left) and quantification (right) of mitochondrial mass (MitoTracker Green FM, Y axis) and mitochondrial potential (MitoTracker Red CMXRos, X axis) in TAMs from Tsc1^fl/flPyMT (n = 7) and CD11c^CreTsc1^fl/flPyMT (n = 7) mice. (D) Representative flow cytometric analysis (left) and quantification (right) of mitochondrial ROS (MitoSOX Red) production in TAMs from Tsc1^fl/flPyMT (n = 9) and CD11c^CreTsc1^fl/flPyMT (n = 9) mice. (E) Representative analysis (left) and quantification (right) of extracellular acidification rate (ECAR) of TAMs from Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT mice. Sequential chemical treatments are indicated (Oligo, oligomycin; 2-DG, 2-Deoxy-D-glucose). (F) Representative analysis (left) and quantification (right) of oxygen consumption rate (OCR) of TAMs from Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT mice. Sequential chemical treatments are indicated (Oligo, oligomycin; FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone; Ant, antimycin A; Rot, rotenone). All data are shown as mean ± SEM, unpaired t-test, two-tailed, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, and n.s. = not significant. See also Figure S6.

Figure 7.. TSC1-deficient TAMs outcompete intratumoral endothelial progenitor cells

(A) Representative images of tumor sections from Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT mice with F4/80 (white), CD31 (red), protein C receptor (PROCR, green), cleaved caspase 3 (CC3, cyan), and DAPI staining (left). Quantification of PROCR⁺ fraction among CD31⁺ endothelial cells by Manders’ colocalization analysis (top right) as well as CD31⁺CC3⁺ events differentiated by PROCR expression (bottom right) in Tsc1^fl/flPyMT and CD11c^CreTsc1^fl/flPyMT tumor tissue sections (n = 10). PROCR⁺CD31⁺CC3⁺ events are marked by hashtag, and PROCR⁻CD31⁺CC3⁺ events are marked by asterisk in images from CD11c^CreTsc1^fl/flPyMT tumors. Scale bars: 50 μm, 10 μm, 50 μm, and 10 μm. (B) PROCR⁺ or PROCR⁻ endothelial cells (ECs) were co-cultured with either wild-type or TSC1-deficient TAMs for 3 days. Representative flow cytometric analysis of ECs and TAMs (left) as well as their numbers (right) were shown. All data are shown as mean ± SEM, unpaired t-test, two-tailed, *: p < 0.05, **: p < 0.01, ****: p < 0.0001, and n.s. = not significant. See also Figure S7.